Program Director/Principal Investigator (Last, First, Middle): PROJECT SUMMARY Currently there is no cure available for HIV/AIDS. Although antiretroviral therapy (ART) is effective in suppressing circulating HIV levels, it does not eliminate the latent and persistent viral reservoirs and the virus rebounds following ART-interruption. Thus, curative therapies are urgently needed to clear the virus from infected people and eliminate the dependence on lifelong ART. Recent clinical success of Chimeric antigen receptor (CAR) T cells in leukemia and lymphoma have demonstrated that redirecting CTL activity of T cells with specific CAR expression can overcome the limitations of autologous CTL functions. This project aims to redirect the natural cytotoxicity of gamma delta (??) T cells against SHIV-infection using CD4-CARs that can recognize infected cells by targeting HIV-Env. The CD4-CAR will be coupled with maC46 expression that will prevent HIV fusion and thereby protect the CAR-?? T cells from getting infected in vivo. Our data in rhesus macaques show readily available ?? T cells in lymph nodes and bone marrow at levels similar to peripheral blood, and are particularly enriched in gut mucosa, liver, spleen, and lungs, all potential reservoir tissues for residual HIV infection during ART. Based on their unique functional properties, including (i) well-documented CTL activity in immunotherapy of cancer, (ii) tissue migration, (iii) innate anti-HIV/SIV CTL/effector functions, and (iv) continued stimulation through gut microbial ligands; we hypothesize that CAR-?? T cells will serve as potent anti-HIV CTLs resistant to HIV infection, and migrate to tissues independent of HIV- stimulation to continually target viral reservoirs. In the R21 phase, we will determine the feasibility of autologous CAR-?? T cell adoptive transfer for migration and persistence in tissues and their in vivo response to SHIV infection in rhesus macaques. In vivo (i) distribution, (ii) proliferation, (iii) persistence; and (iv) ex vivo CTL/cytokine effector functions will be examined in the CD4-CAR-?? T cell-treated animals and compared with the vector control group that receive vector transduced autologous ?? T cells. In the R33 phase, we will determine the feasibility of CAR-?? T cell immunotherapy for targeting viral reservoirs in the clinical setting of SHIV-infected macaques on effective ART. The effects of the adoptively transferred CAR-?? T cells on viral load will be evaluated in lymphoid and mucosal tissue reservoirs. The persistence and homing, CTL activity, selective expansion, and ultimately, the efficacy of adoptive transfer with CD4-CAR-?? T cells will be compared with vector control group. Since rhesus macaque is an important pre-clinical model for both HIV pathogenesis and gene therapy, the evaluation of CAR-?? T cell immunotherapy in rhesus/SHIV model has high translational value. The ability of CAR-?? T cells to control viremia following withdrawal of ART would be a significant advancement in HIV treatment and would strongly promote a new clinical trial for CAR-?? T cell immunotherapy in HIV/AIDS. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page